Both parathyroid hormone (PTH) and mechanical stimulation (MS) have been shown to stimulate net bone formation, in vivo. An early response to both stimuli in cultured osteoblasts is a rapid increase in intracellular Ca2+ ([Ca2+]i) that is mediated by both extracellular Ca2+ entry and intracellular Ca2+ release (iCaR). In this proposal, the principal investigator postulates that PTH lowers the mechanical threshold of osteoblasts by altering the mechanisms involved in modulating [Ca2+]i, thereby promoting net bone formation at more physiologic levels of MS. PTH and MS activate a number of responses in osteoblasts capable of mediating this rise in [Ca2+]i, including mechanosensitive, cation-selective channels (MSCC), IP3-induced iCaR and actin cytoskeletal rearrangement, that could act as sites of convergence for these stimuli. Here, the principal investigator proposes to examine the complex interactions of these responses on [Ca2+]i, and the role they play in gene expression and signal amplification in osteoblasts in response to fluid shear and PTH treatment. In this proposal, the principal investigator will use cell biologic, immunofluorescent staining, patch clamp and [Ca2+]i imaging techniques to examine the interaction of fluid shear and PTH stimulation on [Ca2+]i, expression and production of anabolic markers and paracrine release in osteoblasts. He will focus these studies on channel production and function and iCaR in relation to cellular differentiation and cytoskeletal reorganization. The aims of this proposal are to: (1) determine the interaction of PTH and fluid shear on the expression and production of anabolic markers and paracrine factors associated with bone formation and assess the role of the [Ca2+]i increase in these responses; (2) examine the function of MSCC and Ca2+ channels and iCaR in the [Ca2+]i response to fluid shear and PTH as a function of the differentiated state of the cell; and (3) examine the role of the cytoskeleton in activation and "priming" of channels subjected to PTH and fluid forces. These studies are intended to provide new insight into the mechanisms of transduction of the biophysical signal into osteoblastic function and methods to change this response.